Objective To evaluate the reliability of colony PCR in identifying the recombinant clones selected from phage antibody libraries. Methods The digested pComb3 vector and Lc fragments, Lc library plasmid, and Fd fragments were ligated successively. The ligation product was transformed into Escherichia Coli strain XL 1-blue bacilli by electroporation and thus murine Fab phage antibody library was constructed.The transformed recombinant clones selected randomly from libraries were identified simultaneously by colony PCR, plasmid PCR and restriction enzyme digestion. The identification consistency was analyzed. The interference was excluded by control PCR using the relevant constituents as template. Results The educed library content of Lc library was 1.175?10 6 CFU and the content of Fab antibody library was 1.02?10 6 CFU. Different recombinant percentages were obtained through three different identification methods (the Lc positive recombinant percentages by colony PCR, plasmid PCR and enzyme digestion were 100%, 78% and 78%, respectively; the Fd positive recombinant percentages by three methods were 90%, 66% and 66%, respectively; the dual positive recombinant (Fd and Lc insert simultaneously) percentages by three methods were 90%, 50% and 50%, respectively). A high frequency of false-positives appeared in colony PCR identification. Nonspecific amplification of control PCR was presumably induced by some intracellular components in XL 1-blue bacilli. Conclusion The identification of the recombinant clones selected from phage antibody libraries by colony PCR remains ambiguous. So it is our assertion that the traditional identification methods such as plasmid PCR or enzyme digestion are more accurate and will less false positive results.

Objective To construct human-mouse hybrid Fab phage antibody libraries and to select target Fab genes. Methods With Fd repertoire genes of PBMC from hepatoma patients and the chemeric light chain gene of cFab-HAb18, two hybrid Fab phage antibody libraries(pComb3 and pComb3X) were constructed. The GST fusion and non-fusion HAb18GE were used as antigens to select the target antibodies with the optimized strategies of amplifying and screening. Results When pComb3 displaying library was cultivated at 37℃, the target fragments could not be cut out with the restriction endonucleases from most clones of the library, which was an indication of “recombinant-deletion”. But no “recombinant-deletion” was found in pComb3X displaying library. Because of the serious cross-reaction, the phage-display Fab ELISA could not be used for the selection of positive clones. But positive clones could be identified in the supernatant of lysate with ELISA afterbeing induced by IPTG. Conclusion Through the construction of HuMFab phage antibody libraries and the optimization of screening strategies, we get the desired HuMFab genes for subsequent research.

Objective: To select anti-HAb18G (hepatoma associated antigen) human Fd fragments with guided selection of L chain of chimeric Fab-HAb18. Methods: The human Fd repertories genes were amplified by RT-PCR from PBMC of hepatoma patients, and cloned into the vector pComb3X with chimeric L chain to construct the human-mouse hybrid Fab phage library. HAblSGE, extracellular region of HAblSG, was used as antigen to screen. The positive clones was obtained by ELISA and FCM with p Ⅲ-fusion Fab antibody. The DNA sequences were analyzed. Results: A human-mouse Fab antibody library were constructed with 2?107 PFU. After 6 rounds panning, 7 positive clones were obtained with ELISA and FCM. And sequences of 2 clones with better affinity were same. The CHI belonged to the IgG2 class as the parent Fd, and the variable region belonged to VH3 family. Conclusions: Through the construction of the HuMFab phage antibody library and chemeric L chain-guided selection, we get the available human Fd fragments for subsequent research.

Objective To establish a novel TRFIA for the measurement of heparanase (HPA) in serum samples,and investigate its clinical application.Methods The micro-pore plate wells were first coated with partially recombinant murine anti-human HPA monoclonal antibody.Biotin-labeled recombinant HPA protein was then used to compete with HPA in serum samples,and the prepared europium (III)-labeled streptavidin (Eu3+-SA) was used as signal readout for establishing the BSA-TRFIA assay.Using this assay,the serum HPA levels in healthy subjects (n=32) and tumor patients (n=54) were measured.The results of BSA-TRFIA were compared with those of ELISA.Two-sample t test (or t' test),and linear correlation analysis were used to analyze the data.Results The sensitivity of BSA-TRFIA for measuring HPA was 0.33 ug/L.The CV values for intra-batch and inter-batch were 5.29% and 7.54%,respectively.The average recovery rate was 105.5%.The standard curve range was 0-1 000 ug/L.The serum HPA level measured by the BSA-TRFIA method in healthy subjects was (2.03_+ 1.47) Iug/L.In tumor patients,the HPA level was significantly higher:(22.13_+7.38) ug/L (t'=19.388,P

Objective To evaluate dendritic cell vaccine immunotherapy in postoperative colorectal cancer patients. Methods 40 colorectal cancer patients were divided into two groups. Group A was not treated with the dendritic cell vaccine after chemotherapy, Group B was treated with four courses of autologous dendritic cell vaccine. The level of cytokines was tested before and after the therapy in both groups. DTH were tested after the last course of treatment. Results The levels of cytokines significantly increased in group B after vaccination compared with that in group B before vaccination and that in group A.8 patients were DTH positive in 15 patients that were tested after the vaccination. The PTS (progression-free survival) was 22 months in group B compared with 17 months in group A. The recurrence of the disease was not observed in patients with DTH positive. Conclusion DC vaccine in postoperative colorectal cancer patients improves the immune status and elicits tumor-specific response.

Objective To explore the clinical feature of Kimura disease complicated with nephrotic syndrome in children. Methods The clinical data from 4 children with Kimura disease complicated with nephrotic syndrome were retrospectively analyzed. Results In all of the 4 male children, level of serum IgE was increased but level of eosinophils was not increased in peripheral blood. The renal pathological manifestations were different among them. Two cases had acute tubular injury, one case had pathological changes in repeated renal biopsy, and one case had a little of eosinophils infiltration in renal interstitium. All of them were sensitive to oral hormone treatment. Except that one case had no relapse yet in short follow-up period, the other 3 cases had relapsed and all manifested as positive urinary protein without lymphadenectasis. The 3 cases with recurrence were treated by combined immunosuppressive agents, the palindromia of two cases were reduced which were combined with tacrolimus. Conclusion The Kimura disease complicated with nephrotic syndrome in children has a long course and different renal pathological manifestations. It is sensitive to hormone treatment but easy to relapse, and the treatment should be combined with immunosuppressive therapy. Tacrolimus may have the effect on reducing relapse and delaying the progress of renal pathology.

Objective To investigate and evaluate the changes of T lymphocytes and red cell immunity of peripheral blood in patients with primary hepatocyte carcinoma (PHCC) after radiofrequency ablation(RFA) treatment. Methods The pre- and post- RFA(3d,7d,14d) peripheral blood T lymphocyte subsets(T3,T4,T8,T4/T8) and red cell immunity (RBC C3 receptor flower and RBC-immuocomplex formation rate) were investigated in 120 patients with PHCC treated by RFA. Results On 7d, 14d after RFA, T3, T4 lymphocytes and T4/T8 were higher than those on preoperative day significantly(P

OBJECTIVETo compare the short-term efficacy between totally laparoscopic distal gastrectomy(TLDG) with delta-shaped anastomosis (DS) and laparoscopic-assisted distal gastrectomy (LADG) with BrillrothI ( anastomosis (BI(), and to evaluate the application of DS.

METHODSBetween March 2013 and February 2014, 50 patients underwent TLDG with DS using linear staplers, and 43 patients underwent LADG with BI( using circular staplers. Clinical features and short-term efficacy of the two groups were analyzed retrospectively.

RESULTSThere were no significant differences between the two groups in terms of demographic indicators, operation time, intraoperative blood loss, number of removal lymph node, time to first flatus, incidence of complication and postoperative discharge day(all P>0.05). First-day postoperative pain was milder (3.1 ± 1.0 vs. 4.6 ± 1.4), and operative incision was shorter [(3.4 ± 0.4) cm vs. (6.9 ± 0.8) cm] significantly in TLDG with DS group(P<0.05).

CONCLUSIONTLDG with DS is safe and feasible for patients with gastric cancer, and has more advantages in cosmetic and comfort level than LADG with BI.

Anastomosis, Surgical ; Gastrectomy ; Humans ; Laparoscopy ; Lymph Node Excision ; Operative Time ; Postoperative Period ; Retrospective Studies ; Stomach ; surgery ; Stomach Neoplasms

OBJECTIVE： To establish HPLC fingerprints of Ligusticum chuanxiong decoction pieces， and to conduct cluster analysis and PLS-DA analysis. METHODS： HPLC method was adopted. The determination was performed on Waters Symmetry C18 column with mobile phase consisted of acetonitrile-0.5％ acetic acid solution （gradient elution） at the flow rate of 1 mL/min. The detection wavelength was set at 254 nm， and the column temperature was 30 ℃. The sample size was 10 μL. Using ligustilide as control， HPLC chromatograms of 21 batches of samples （S1-S20） were determined. The similarity evaluation was conducted by using Similarity Evaluation System for Chromatographic Fingerprint of TCM （2012 edition） to determine common peak. Cluster analysis was conducted by using SPSS 19.0 software and PLS-DA was used to distinguish the samples. RESULTS： There were 25 common peaks in HPLC chromatograms for 21 batches of samples， and 9 common peaks were identified. The similarity of samples was between 0.768-0.989， and the similarity of base and traditional medicinal part samples （S1-S10） were more than 0.970. The 21 batches of samples were clustered into 3 categories， in which S1-S10 were category Ⅰ； S15-S16， S19-S20 were category Ⅱ； other were category Ⅲ. By PLS-DA analysis， 11 classification markers were identified as well as 5 chromatogram peaks were identified， such as ferulic acid， pine cyperyl ferulate， n-butyl phthalide， ligustilide， ligustilide A， which could be used to distinguish base and non-markted samples （S1-S10） from marketed and non-base samples （S11-S21）， which were consistent with the results of cluster analysis. CONCLUSIONS： Established fingerprint， cluster analysis and PLS-DA analysis can provide reference for quality evaluation of L. chuanxiong decoction pieces.

Femoral neck fracture (FNF) in the elderly patients is currently a major health challenge worldwide, with excessive consumption of medical resources, high incidence of complications as well as suboptimal outcome and prognosis. Hip joint arthroplasty (HJA) has been the mainstream treatment for FNF in the elderly, but the conventional surgical approaches and techniques are still confronted with a series of bottlenecks such as dislocation, limp and limb length discrepancy. In recent years, direct anterior approach (DAA) for HJA (DAA-HJA) has been a major new choice in the field of joint replacement, which achieves improved clinical effectiveness of HJA in the treatment of elderly FNF, due to the fact that DAA approach involves the neuromuscular interface and accords with the idea of soft tissue retention and enhanced recovery after surgery. However, there is still a lack of unified understanding of standard technique and procedure of DAA-HJA in the treatment of elderly FNF. Therefore, relevant experts from the Hip Joint Group of Chinese Orthopedics Association of Chinese Medical Association, Youth Arthrology Group of Orthopedic Committee of PLA, Orthopedic Committee of Chongqing Medical Association, Branch of Orthopedic Surgeons of Chongqing Medical Doctor Association and Sport Medicine Committee of Chongqing Medical Association were organized to formulate the " Chinese expert consensus on the technical standard of direct anterior hip arthroplasty for elderly femoral neck fracture ( version 2023)" based on evidence-based medicine. This consensus mainly proposed 13 recommendations covering indications, surgical plans, prosthesis selections, surgical techniques and processes, and postoperative management of DAA-HJA in elderly patients with FNF, aiming to promote standardized, systematic and patient-specific diagnosis and treatment to improve the functional prognosis of the patients.